microscopy fits needs of researcher, type of specimen being viewed brightfield brightfield darkfield...

Microscopy fits needs of Microscopy fits needs of researcher, type of researcher, type of

specimen being viewedspecimen being viewed

BrightfieldBrightfield DarkfieldDarkfield DIC/NomarskiDIC/Nomarski UltrastructuralUltrastructural Dimensional microscopy (SEM and Dimensional microscopy (SEM and

Confocal)Confocal) Simple magnification Simple magnification

Resolution Depends Upon Resolution Depends Upon Magnification, Type of Scope, Magnification, Type of Scope, Specimen Fixation, Thickness, Specimen Fixation, Thickness,

StainingStaining Staining types – GeneralStaining types – General

Acid stains (eg., eosin, orange G, aniline Acid stains (eg., eosin, orange G, aniline blue), blue),

Basic stains (Methyl green, methylene blue)Basic stains (Methyl green, methylene blue) Mordants (Hematoxylin, a generally basic Mordants (Hematoxylin, a generally basic

dye, is a mixture of hematoxylin and mordant dye, is a mixture of hematoxylin and mordant (link b/w dye and tissue) generally used in (link b/w dye and tissue) generally used in connection with acidic dyes to provide connection with acidic dyes to provide contrast and staining of cellular componentscontrast and staining of cellular components

Specific tissue component stains (eg., PAS Specific tissue component stains (eg., PAS for carbohydrates)for carbohydrates)

Staining Types - SpecificStaining Types - Specific ImmunohistochemistryImmunohistochemistry Enzyme HistochemistryEnzyme Histochemistry AutoradiographyAutoradiography

Resolution, cont’dResolution, cont’d

Microscope Slide

Primary antibodySecondary Antibody

Protein in tissue

Fluorescent or enzymatic label

Immunohistochemistry

Enzyme in cells of tissue

Substrate

Enzyme Histochemistry

AutoradiographyAutoradiography Incorporation of label into DNAIncorporation of label into DNA

Injection of labelled tracer into animal or cellInjection of labelled tracer into animal or cell Removal of tissue, fixation, sectioningRemoval of tissue, fixation, sectioning Exposure of section to photographic emulsionExposure of section to photographic emulsion Emission of radioactive tracer reacts with Emission of radioactive tracer reacts with

emulsionemulsion Emulsion is developed, silver grains denote Emulsion is developed, silver grains denote

label locationlabel location Counterstain and viewCounterstain and view

Areas of label indicative of cellular Areas of label indicative of cellular process (eg., making DNA, protein) being process (eg., making DNA, protein) being examinedexamined

Guess the Technique

- control + control)

- control + control